The present invention relates to a method for staining a fecal specimen preserved using a non-mercury based fixative. More particularly, the invention provides a method which utilizes a stain which is a combination of iron hematoxylin stain and trichrome stain for staining fecal specimens preserved in non-mercury based fixatives.
The most widely used method for detecting and identifying gastrointestinal parasites is the microscopic examination of fecal specimens in the laboratory. Ordinarily, a part of this microscopic examination involves staining the specimen in order to enable the clinician to differentiate between organisms which, unstained, are very difficult to distinguish with accuracy.
Because parasites are fragile and can change or deteriorate in the time which elapses before they are examined, specimens must be examined immediately or must be preserved with a suitable fixative. Timely collection and transportation of fresh stool specimens to the laboratory cannot always be assured and workload conditions and priorities in clinical laboratories frequently do not permit immediate examination of fresh specimens. Procedures such as incubation, refrigeration or freezing do not guarantee the recovery of all diagnostic stages of all parasites. Therefore, to prevent the degradation and loss of stool parasites from a specimen, fixatives are used to preserve the specimen.
Conventionally, mercury based fixatives have been used to preserve stool specimens for staining and microscopic parasitological examination. Stool parasites that have been preserved in mercury based fixatives provide good definition of the parasite upon staining. However, mercury based fixatives have a number of disadvantages associated with them that limit their use and have prompted laboratories to adopt non-mercury fixatives. In particular, the use and disposal of mercury is regulated under federal and state environmental laws.
Non-mercury based fixatives for parasitological examination have been developed in recent years in response to the health and environmental concerns associated with mercury based preservatives. These fixatives typically include non-mercury metals zinc or copper together with polymers such as polyvinyl alcohol. Specimens prepared in non-mercury based fixatives do not stain with the intensity, contrast and resolution compared to stained stool specimens which have been preserved in mercury based fixatives. This makes it more difficult and tedious to detect and identify the parasites. Accordingly, there is a need for a methodology for staining non-mercury fixed specimens which provides improved contrast and intensity.
Trichrome stain and iron hematoxylin stain are two stains frequently used in diagnostic parasitology. The trichrome stain works excellently with mercury fixed fecal specimens but does not work nearly as well when used with non-mercury fixatives. As a result, attempts have been made to find better stains for specimens fixed with non-mercury fixatives.
One prior art attempt to develop an improved stain is described in U.S. Pat. No. 4,666,699 to Slifkin, which teaches a stain-fixative composition for enteric parasites for use with mercury-free and formalin-free fixatives. The composition contains Ponceau S Stain, Chlorazol Fast Pink BK Stain, Trypan Blue Stain, dimethyl sulfoxide and a non-mercury, non-formalin based fixative.
There continues to be a need for a method for staining stool parasite specimens which have been preserved in non-mercury based fixatives that produces significantly better overall staining quality, intensity and contrast.
The present invention is based on the discovery that iron hematoxylin stain and trichrome stain can be mixed and used effectively to stain fecal specimens preserved with non-mercury fixatives. Surprisingly, these two stains are not incompatible, they do not interact, they are not redundant, they intensify each other and in combination they stain fecal specimens with an intensity and resolution that is significantly better than can be achieved using either stain alone or in sequence. Whereas trichrome stain and hematoxylin stain when used alone with non-mercury fixatives give a low contrast microscope image which is difficult and time consuming to view and evaluate, when combined they yield an image which is intense and much easier to read and evaluate. The method of the present invention facilitates more accurate and precise detection of stool parasites by producing significantly better overall staining quality, intensity and contrast than previous methods for staining stool parasites preserved in non-mercury based fixatives.